Background The high concentrated thrombin time (hcTT), a thrombin time modified by increasing the thrombin concentration, is a possible alternative assay to activated partial thromboplastin time (aPTT) in unfractionated heparin (UFH) monitoring. This study aimed to determine the optimal thrombin concentration used in the hcTT assay for UFH monitoring. Methods A total of 30 blood samples obtained from healthy volunteers were included in this study. Thrombin concentrations of 10.0, 15.0, 20.0, and 25.0 IU/ml were used in the hcTT assay. The consistency between the hcTT and anti‐FXa assays was evaluated. To validate the hcTT assay, linearity, repeatability, reproducibility, and diagnostic performance of the assay were assessed. Results The hcTT assay using thrombin concentration of 15.0 IU/ml showed a strong correlation to the anti‐FXa assay with R2 of 0.72 and the Spearman's correlation coefficient (rs) of 0.97 (95% CI, 0.96–0.98). Within‐run and day‐to‐day run variabilities of the assay were satisfactory (all coefficients of variation <10%). We found an excellent correlation between the results which were measured using different reagents with intra‐ or inter‐laboratory instruments. Notably, as compared to the aPTT assay, the hcTT assay showed a significantly better performance in identifying the samples which contain UFH at the supratherapeutic level, with an AUC of 0.97 vs. 0.91, p = 0.049. Conclusion The hcTT assay can be used as an alternative assay for UFH therapy monitoring. A further study using clinical samples is recommended to confirm the appropriateness of the hcTT assay for clinical application.